The small GTPase Rab22 interacts with EEA1 and controls endosomal membrane trafficking

2002 ◽  
Vol 115 (5) ◽  
pp. 899-911 ◽  
Author(s):  
Maria Kauppi ◽  
Anne Simonsen ◽  
Bjørn Bremnes ◽  
Amandio Vieira ◽  
Judy Callaghan ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Hela Cells ◽  
Membrane Trafficking ◽  
Fluid Phase ◽  
Small Gtpase ◽  
Wild Type ◽  
Dynamic Interactions ◽  
Recycling Endosomes ◽  
Endosomal Membrane ◽  
Gtpase Cycle

Rab22a is a small GTPase that is expressed ubiquitously in mammalian tissues and displays the highest sequence homology to Rab5. In BHK-21 cells,overexpression of the wild-type Rab22a caused formation of abnormally large vacuole-like structures containing the early-endosomal antigen EEA1 but not Rab11, a marker of recycling endosomes or the late-endosomal/lysosomal markers LAMP-1 and lyso-bis-phosphatidic acid. In HeLa cells, overexpressed Rab22a was found on smaller EEA1-positive endosomes, but a portion of the protein was also found in the Golgi complex. Using the yeast two-hybrid system and a biochemical pull-down assay, the GTP-bound form of Rab22a was found to interact with the N-terminus of EEA1. In HeLa cells overexpressing Rab22a or its mutants affected in the GTPase cycle, no significant changes were observed in the uptake of Alexa-transferrin. However, the GTPase-deficient Rab22a Q64L mutant caused a redistribution of transferrin-positive endosomes to the leading edges of cells and a fragmentation of the Golgi complex. In BHK cells,the Q64L mutant caused the accumulation of a fluid phase marker,TRITC-dextran, and a lysosomal hydrolase, aspartylglucosaminidase, in abnormal vacuole-like structures that contained both early and late endosome markers. Both the wild-type Rab22a and the Q64L mutant were found to interfere with the degradation of EGF. These results suggest that Rab22a may regulate the dynamic interactions of endosomal compartments and it may be involved in the communication between the biosynthetic and early endocytic pathways.

scholarly journals Connecdenn 3/DENND1C binds actin linking Rab35 activation to the actin cytoskeleton

2012 ◽  
Vol 23 (1) ◽  
pp. 163-175 ◽  
Author(s):  
Andrea L. Marat ◽  
Maria S. Ioannou ◽  
Peter S. McPherson
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Small Gtpase ◽  
Actin Binding ◽  
Binding Motif ◽  
Endosomal Trafficking ◽  
Nucleotide Exchange ◽  
Endosomal Membrane ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

The small GTPase Rab35 regulates endosomal membrane trafficking but also recruits effectors that modulate actin assembly and organization. Differentially expressed in normal and neoplastic cells (DENN)–domain proteins are a newly identified class of Rab guanine-nucleotide exchange factors (GEFs) that are grouped into eight families, each activating a common Rab. The members of one family, connecdenn 1–3/DENND1A–C, are all GEFs for Rab35. Why Rab35 requires multiple GEFs is unknown. We demonstrate that connecdenn 3 uses a unique C-terminal motif, a feature not found in connecdenn 1 or 2, to directly bind actin. This interaction couples Rab35 activation to the actin cytoskeleton, resulting in dramatic changes in cell shape, notably the formation of protrusive membrane extensions. These alterations are specific to Rab35 activated by connecdenn 3 and require both the actin-binding motif and N-terminal DENN domain, which harbors the GEF activity. It was previously demonstrated that activated Rab35 recruits the actin-bundling protein fascin to actin, but the relevant GEF for this activity was unknown. We demonstrate that connecdenn 3 and Rab35 colocalize with fascin and actin filaments, suggesting that connecdenn 3 is the relevant GEF. Thus, whereas connecdenn 1 and 2 activate Rab35 for endosomal trafficking, connecdenn 3 uniquely activates Rab35 for its role in actin regulation.

scholarly journals Mycobacterium tuberculosis and Legionella pneumophila Phagosomes Exhibit Arrested Maturation despite Acquisition of Rab7

2000 ◽  
Vol 68 (9) ◽  
pp. 5154-5166 ◽  
Author(s):  
Daniel L. Clemens ◽  
Bai-Yu Lee ◽  
Marcus A. Horwitz
Keyword(s):  
Hela Cells ◽  
Legionella Pneumophila ◽  
Small Gtpase ◽  
Endocytic Pathway ◽  
Membrane Glycoprotein ◽  
Mutant Form ◽  
Wild Type ◽  
Vesicular Traffic ◽  
Inert Particles ◽  
Constitutively Active

ABSTRACT Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. Phagosomes containing inert particles have also been shown to transiently acquire Rab7 as they mature. Disruption in the pathway prior to the acquisition of Rab7 has been suggested as playing a role in the altered maturation of Mycobacterium bovis BCG phagosomes. As a first step to determine whether disruption in the delivery or function of Rab7 could play a role in the altered maturation of Legionella pneumophila and M. tuberculosis phagosomes, we have examined the distribution of wild-type Rab7 and the GTPase-deficient, constitutively active mutant form of Rab7 in HeLa cells infected withL. pneumophila or M. tuberculosis. We have found that the majority of L. pneumophila and M. tuberculosis phagosomes acquire relatively abundant staining for Rab7 and for the constitutively active mutant Rab7 in HeLa cells that overexpress these proteins. Nevertheless, despite acquisition of wild-type or constitutively active Rab7, both the L. pneumophila and the M. tuberculosis phagosomes continue to exhibit altered maturation as manifested by a failure to acquire lysosome-associated membrane glycoprotein 1. These results demonstrate that L. pneumophila and M. tuberculosis phagosomes have receptors for Rab7 and that the altered maturation of these phagosomes is not due to a failure to acquire Rab7.

scholarly journals Dynein-mediated Vesicle Transport Controls Intracellular Salmonella Replication

2004 ◽  
Vol 15 (6) ◽  
pp. 2954-2964 ◽  
Author(s):  
Marije Marsman ◽  
Ingrid Jordens ◽  
Coen Kuijl ◽  
Lennert Janssen ◽  
Jacques Neefjes
Keyword(s):  
Hela Cells ◽  
Small Gtpase ◽  
Vesicle Transport ◽  
Endocytic Pathway ◽  
Rab Gtpases ◽  
Intracellular Replication ◽  
Motor Complex ◽  
Late Endosomes ◽  
Membrane Bound ◽  
Gtpase Cycle

Salmonella typhimurium survives and replicates intracellular in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). In HeLa cells, the SCV matures through interactions with the endocytic pathway, but Salmonella avoids fusion with mature lysosomes. The exact mechanism of the inhibition of phagolysosomal fusion is not understood. Rab GTPases control several proteins involved in membrane fusion and vesicular transport. The small GTPase Rab7 regulates the transport of and fusion between late endosomes and lysosomes and associates with the SCV. We show that the Rab7 GTPase cycle is not affected on the SCV. We then manipulated a pathway downstream of the small GTPase Rab7 in HeLa cells infected with Salmonella. Expression of the Rab7 effector RILP induces recruitment of the dynein/dynactin motor complex to the SCV. Subsequently, SCV fuse with lysosomes. As a result, the intracellular replication of Salmonella is inhibited. Activation of dynein-mediated vesicle transport can thus control intracellular survival of Salmonella.

scholarly journals Regulation of Protein Transport from the Golgi Complex to the Endoplasmic Reticulum by CDC42 and N-WASP

2002 ◽  
Vol 13 (3) ◽  
pp. 866-879 ◽  
Author(s):  
Ana Luna ◽  
Olga B. Matas ◽  
José Angel Martı́nez-Menárguez ◽  
Eugenia Mato ◽  
Juan M. Durán ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Mammalian Cells ◽  
Protein Transport ◽  
Retrograde Transport ◽  
Small Gtpase ◽  
Actin Dynamics ◽  
Mutant Form ◽  
Dependent Manner ◽  
Wild Type ◽  
Kdel Receptor

Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and noncoated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgi-to-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(ΔWA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-N-WASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP–dependent manner.

scholarly journals Deviant Expression of Rab5 on Phagosomes Containing the Intracellular Pathogens Mycobacterium tuberculosis andLegionella pneumophila Is Associated with Altered Phagosomal Fate

2000 ◽  
Vol 68 (5) ◽  
pp. 2671-2684 ◽  
Author(s):  
Daniel L. Clemens ◽  
Bai-Yu Lee ◽  
Marcus A. Horwitz
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hela Cells ◽  
Legionella Pneumophila ◽  
Critical Role ◽  
Small Gtpase ◽  
Biologically Active ◽  
Host Cells ◽  
Molecular Characteristics ◽  
Wild Type ◽  
Immunogold Staining

ABSTRACT The intracellular human pathogens Legionella pneumophila and Mycobacterium tuberculosis reside in altered phagosomes that do not fuse with lysosomes and are only mildly acidified. The L. pneumophila phagosome exists completely outside the endolysosomal pathway, and the M. tuberculosisphagosome displays a maturational arrest at an early endosomal stage along this pathway. Rab5 plays a critical role in regulating membrane trafficking involving endosomes and phagosomes. To determine whether an alteration in the function or delivery of Rab5 could play a role in the aberrant development of L. pneumophila and M. tuberculosis phagosomes, we have examined the distribution of the small GTPase, Rab5c, in infected HeLa cells overexpressing Rab5c. Both pathogens formed phagosomes in HeLa cells with molecular characteristics similar to their phagosomes in human macrophages and multiplied in these host cells. Phagosomes containing virulent wild-type L. pneumophila never acquired immunogold staining for Rab5c, whereas phagosomes containing an avirulent mutant L. pneumophila (which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast, M. tuberculosis phagosomes exhibited abundant staining for Rab5c throughout its life cycle. To verify that the overexpressed, recombinant Rab5c observed on the bacterial phagosomes was biologically active, we examined the phagosomes in HeLa cells expressing Rab5c Q79L, a fusion-promoting mutant. Such HeLa cells formed giant vacuoles, and after incubation with various particles, the giant vacuoles acquired large numbers of latex beads, M. tuberculosis, and avirulent L. pneumophila but not wild-type L. pneumophila, which consistently remained in tight phagosomes that did not fuse with the giant vacuoles. These results indicate that whereas Rab5 is absent from wild-type L. pneumophilaphagosomes, functional Rab5 persists on M. tuberculosisphagosomes. The absence of Rab5 on the L. pneumophilaphagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on the M. tuberculosis phagosomes may enable the phagosome to retard its own maturation at an early endosomal stage.

Arfs, phosphoinositides and membrane traffic

2005 ◽  
Vol 33 (6) ◽  
pp. 1276-1278 ◽  
Author(s):  
J.G. Donaldson
Keyword(s):  
Golgi Complex ◽  
Membrane Trafficking ◽  
Membrane Lipids ◽  
Membrane Traffic ◽  
Type I ◽  
Coat Proteins ◽  
Endosomal Membrane ◽  
Membrane Surfaces ◽  
Phosphatidylinositol 4 ◽  
Cytoskeletal Elements

Arf (ADP-ribosylation factor) GTP-binding proteins function in cells to regulate membrane traffic and structure. Arfs accomplish this task through modification of membrane lipids and the recruitment of proteins, including coat proteins and actin, to membrane surfaces. Arf1 and Arf6 are the most divergent and most studied human Arf proteins that localize predominantly to the Golgi complex and plasma membrane respectively. We have been studying the targeting of Arf1 and Arf6 to these specific compartments and the common and divergent activities that they exert on these membranes. We have found that Arf6 acts through activation of type I phosphatidylinositol 4-phosphate 5-kinases to generate phosphatidylinositol 4,5-bisphosphate and that this activity is instrumental in facilitating the actin cytoskeletal rearrangements and alterations in endosomal membrane trafficking observed with increased Arf6 activation. Arf1 can also stimulate the activity of phosphatidylinositol kinases and recruit coat proteins and actin cytoskeletal elements to the Golgi complex.

scholarly journals Inactivation of lmpA, Encoding a LIMPII-related Endosomal Protein, Suppresses the Internalization and Endosomal Trafficking Defects in Profilin-null Mutants

2000 ◽  
Vol 11 (6) ◽  
pp. 2019-2031 ◽  
Author(s):  
Lesly Temesvari ◽  
Linyi Zhang ◽  
Brent Fodera ◽  
Klaus-Peter Janssen ◽  
Michael Schleicher ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Fluid Phase ◽  
Actin Binding ◽  
Endosomal Trafficking ◽  
Wild Type ◽  
Gene Encoding ◽  
Biochemical Assays ◽  
Endosomal Pathway ◽  
Trafficking Defects

Profilin is a key phosphoinositide and actin-binding protein connecting and coordinating changes in signal transduction pathways with alterations in the actin cytoskeleton. Using biochemical assays and microscopic approaches, we demonstrate that profilin-null cells are defective in macropinocytosis, fluid phase efflux, and secretion of lysosomal enzymes but are unexpectedly more efficient in phagocytosis than wild-type cells. Disruption of the lmpA gene encoding a protein (DdLIMP) belonging to the CD36/LIMPII family suppressed, to different degrees, most of the profilin-minus defects, including the increase in F-actin, but did not rescue the secretion defect. Immunofluorescence microscopy indicated that DdLIMP, which is also capable of binding phosphoinositides, was associated with macropinosomes but was not detected in the plasma membrane. Also, inactivation of the lmpA gene in wild-type strains resulted in defects in macropinocytosis and fluid phase efflux but not in phagocytosis. These results suggest an important role for profilin in regulating the internalization of fluid and particles and the movement of material along the endosomal pathway; they also demonstrate a functional interaction between profilin and DdLIMP that may connect phosphoinositide-based signaling through the actin cytoskeleton with endolysosomal membrane trafficking events.

scholarly journals The phospholipid flippase ATP9A is required for the recycling pathway from the endosomes to the plasma membrane

2016 ◽  
Vol 27 (24) ◽  
pp. 3883-3893 ◽  
Author(s):  
Yoshiki Tanaka ◽  
Natsuki Ono ◽  
Takahiro Shima ◽  
Gaku Tanaka ◽  
Yohei Katoh ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Golgi Complex ◽  
Membrane Trafficking ◽  
Glucose Transporter ◽  
Glucose Transporter 1 ◽  
Recycling Endosomes ◽  
Type Iv ◽  
Late Endosomes ◽  
P Type

Type IV P-type ATPases (P4-ATPases) are phospholipid flippases that translocate phospholipids from the exoplasmic (or luminal) to the cytoplasmic leaflet of lipid bilayers. In Saccharomyces cerevisiae, P4-ATPases are localized to specific subcellular compartments and play roles in compartment-mediated membrane trafficking; however, roles of mammalian P4-ATPases in membrane trafficking are poorly understood. We previously reported that ATP9A, one of 14 human P4-ATPases, is localized to endosomal compartments and the Golgi complex. In this study, we found that ATP9A is localized to phosphatidylserine (PS)-positive early and recycling endosomes, but not late endosomes, in HeLa cells. Depletion of ATP9A delayed the recycling of transferrin from endosomes to the plasma membrane, although it did not affect the morphology of endosomal structures. Moreover, depletion of ATP9A caused accumulation of glucose transporter 1 in endosomes, probably by inhibiting their recycling. By contrast, depletion of ATP9A affected neither the early/late endosomal transport and degradation of epidermal growth factor (EGF) nor the transport of Shiga toxin B fragment from early/recycling endosomes to the Golgi complex. Therefore ATP9A plays a crucial role in recycling from endosomes to the plasma membrane.

scholarly journals The WASp-like protein Scar regulates macropinocytosis, phagocytosis and endosomal membrane flow in Dictyostelium

2001 ◽  
Vol 114 (14) ◽  
pp. 2673-2683
Author(s):  
David J. Seastone ◽  
Ed Harris ◽  
Lesly A. Temesvari ◽  
James E. Bear ◽  
Charles L. Saxe ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Line ◽  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Fluid Phase ◽  
Dictyostelium Cell ◽  
Physical Interaction ◽  
Developmental Defect ◽  
Membrane Flow ◽  
Endosomal Membrane

Scar, a member of the WASp protein family, was discovered in Dictyostelium discoideum during a genetic screen for second-site mutations that suppressed a developmental defect. Disruption of the scar gene reduced the levels of cellular F-actin by 50%. To investigate the role of Scar in endocytosis, phagocytosis and endocytic membrane trafficking, processes that depend on actin polymerization, we have analyzed a Dictyostelium cell line that is genetically null for Scar. Rates of fluid phase macropinocytosis and phagocytosis are significantly reduced in the scar- cell-line. In addition, exocytosis of fluid phase is delayed in these cells and movement of fluid phase from lysosomes to post-lysosomes is also delayed. Inhibition of actin polymerization with cytochalasin A resulted in similar phenotypes, suggesting that Scar-mediated polymerization of the actin cytoskeleton was important in the regulation of these processes. Supporting this conclusion, fluorescence microscopy revealed that some endo-lysosomes were ringed with F-actin in control cells but no F-actin was detected associated with endo-lysosomes in Scar null cells. Disruption of the two genes encoding the actin monomer sequestering protein profilin in wild-type cells causes defects in the rate of pinocytosis and fluid phase efflux. Consistent with a predicted physical interaction between Scar and profilin, disrupting the scar gene in the profilin null background results in greater decreases in the rate of fluid phase internalization and fluid phase release compared to either mutant alone. Taken together, these data support a model in which Scar and profilin functionally interact to regulate internalization of fluid and particles and later steps in the endosomal pathway, probably through regulation of actin cytoskeleton polymerization.

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